Project Summary/Abstract The proteolytic release of ligands of the epidermal growth factor-receptor (EGFR) and the inflammatory cytokine tumor necrosis factor-a (TNFa) is tightly regulated by the cell surface metalloprotease ADAM17 (A disintegrin and metalloproteinase 17). ADAM17-dependent activation of the EGFR is essential for normal heart, bone, skin and eye development, and for the maintenance of the skin and intestinal barrier during adulthood, but can also contribute to the development cancer. Genetic studies in mice have revealed that ADAM17 plays a non- redundant role in these pathways, highlighting the physiological and pathological importance of ADAM17. Mice lacking ADAM17 have impaired EGFR signaling and phenotypically resemble mice lacking the EGFR in their perinatal lethality, open eyes at birth and heart valve defects. Moreover, mice lacking ADAM17 in immune cells are protected from septic shock and TNFa-dependent pathologies such as inflammatory arthritis. A hallmark feature of ADAM17 is its ability to rapidly turn on in response to many physiological stimuli. This rapid activation occurs through a mechanism that requires its transmembrane domain, but not its cytoplasmic domain. The transmembrane domain of ADAM17 interacts with the inactive Rhomboid proteins 1 and 2 (iRhom1/2) to support key aspects of ADAM17 regulation such as maturation, catalytic activity, and substrate selectivity. However, the functional relevance of the ADAM17 cytoplasmic domain has remained elusive. This is in part because overexpressing ADAM17 in cells mainly gives rise to the inactive pro-ADAM17, but little or no mature-ADAM17. In order to circumvent this overexpression artifact, I have characterized a mouse line in which the ADAM17 cytoplasmic domain was truncated at the endogenous ADAM17 locus using CRISPR/Cas9. This truncation resulted in strongly reduced ADAM17 protein levels and a partial loss of function phenotype. This is the first genetic evidence, to our knowledge, that the ADAM17 cytoplasmic domain is functionally relevant and may play a role in regulating the levels of ADAM17. Furthermore, cell-biological data show that the ADAM17 cytoplasmic domain is required for ADAM17/iRhom2-dependent substrate release, suggesting that the ADAM17 cytoplasmic domain is a key mediator of ADAM17/iRhom2 interactions. My goal is to define the cytoplasmic sequences in ADAM17 that control its levels and identify ADAM17 cytoplasmic domain interacting molecules that mediate ADAM17 stability. I will also define the ADAM17 cytoplasmic domain sequence required for ADAM17/iRhom2- dependent catalytic activity. These studies promise to provide new and exciting insights into the regulation of ADAM17 by its cytoplasmic domain. Understanding how the ADAM17 cytoplasmic domain regulates ADAM17 proteins levels offers the possibility of identifying novel therapeutic targets for EGFR-dependent cancers and TNFa-dependent autoimmune disorders.